Rat liver glyoxalase II has been purified to homogeneity by a rapid, two-step procedure involving affinity chromatography on S-carbobenzoxyglutathione coupled to Sepharose 4B. The purified enzyme gives a major band corresponding to 30,000 daltons (monomer) and a minor band corresponding to 120,000 daltons (tetramer) with sodium dodecylsulfate polyacrylamide gel electrophoresis. Evidence is given for the interconversion of the monomer and tetramer forms of glyoxalase II via the dimer and the trimer.